For studying cell biology, a number of techniques are available for detecting, measuring, and following almost any chosen molecule in a cell. Light-microscope techniques are vastly used for observing cells. Cells that have been fixed and stained can be studied in a conventional light microscope, while fluorescent dyes coupled to antibodies can be used to locate specific molecules in cells using a fluorescent microscope. The confocal scanning microscope provides thin optical sections and can be used to reconstruct a three-dimensional image. Three-dimensional views of the surfaces of cells and tissues can be obtained by scanning electron microscopy, while the interior of membrane and cells can be visualised by freeze-fracture and freeze-etch electron microscopy, respectively.
The classical methods of microscopy give good views of cell architecture, but they provide little information about cell chemistry. In cell biology it is often important to determine the quantities of specific molecules and to know where they are in the cell and how their level or location changes in response to extracellular or intracellular signals. The molecules of interest range from small inorganic ions, such as Ca2+ or H+, to large molecules, such as specific proteins, RNAs, or DNA. Sensitive methods have been developed for assaying each of these types of molecules, as well as for following the dynamic behaviour of many of them in living cells. One particular method is the introduction of probes into living cells in order to monitor the chemical conditions within the cell.
Certain probes interestingly consist of fluorogenic or fluorescent dyes coupled to a blocking group thus forming an enzyme substrate. These probes are cleavable by enzymes, and yield fluorescent dye precipitates, which may be detected by fluorescence microscopy in a highly sensitive fashion.
Amongst different enzyme substrates, hydrolase substrates, i.e. substrates recognized by hydrolases, have been conveniently used in assaying cell biology. Hydrolases are enzymes found in a broad variety of organisms, including bacteria, yeast and higher animals and plants. They act by catalyzing a hydrolysis reaction. Thus, they break down a compound (i.e., the substrate) by cleaving a covalent bond in the compound and inserting a water molecule across the bond. Hydrolase enzymes include those that act on ester bonds, on peptide bonds, on carbon-nitrogen bonds other than peptide bonds, on glycoside bonds, on ether bonds, and on acid anhydrides, among others. Esterases, lipases, peptidases, glycosylases, such as glycosidases, phosphatases, sulfatases, nucleases, exonucleases, endonucleases, are typical exponents of this type of enzymes.
Hydrolases may be used in enzyme labelled fluorescence assays, where the fluorescence mechanism is used to detect enzyme activity. Enzyme labelled fluorescence assays are based on the enzymatic conversion of specific substrates into the corresponding fluorescent precipitates. These substrates may also be refereed to as “fluorogenic substrates” as they are able to be cleaved and yield fluorescent precipitates upon enzymatic action.
As such, certain fluorogenic β-D-glucuronidase or β-D-galactosidase substrates are known. For instance, the use of the β-D-galactosidase (GAL) enzyme and its fluorogenic substrate 4-methylumbelliferyl β-D-galactoside has been cited in the literature as an example (Ishikawa E., Imagawa M., & Hashids S., “Ultrasensitive Enzyme Immunoassay Using Fluorogenic, Lumenogenic, Radioactive and Related Substrates and Factors to Limit Sensitivity”, J. Biochem 73, 1319-1321, 1973). Also the use of the β-D-glucuronidase (GUS) enzyme and its fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide has been described (Jefferson R. A. “Assaying Chimeric Genes in Plants: The GUS Gene Fusion System”, Plant Mol. Biol. Rep. 5, 387-405, 1987; Jefferson R. A. “The GUS Reporter Gene System”, Nature, 342, 837-838, 1989).
Also fluorogenic substrates that are made from a class of fluorophores, generally including quinazolinones (quinazolones), benzimidazoles, benzothiazoles, benzoxazoles, quinolines, indolines, and phenanthridines, have been described. These substrates can be enzymatically converted to a detectable phenolic product, e.g. formation of a soluble coloured or fluorescent product or formation of a precipitate. For example U.S. Pat. No. 5,316,906 and U.S. Pat. No. 5,433,986 describe this kind of substrates which consist of substances coupled with phosphate, sulfate or sugar groups and which form a highly fluorescent precipitate upon reaction with the appropriate enzyme. Both US patents also mention the use of these fluorogenic substrates to detect and study enzyme activity. Particular examples of this kind of substrates include the ELF97® β-D-galactosidase substrate (ELF97® β-D-galacto-pyranoside) (1) and the ELF97® β-D-glucuronidase substrate (ELF97® β-D-glucuronide) (2) that have been developed and awe commercially available.

These ELF97® β-D-galactosidase and ELF97® β-D-glucuronidase substrates are non-fluorescent but can react with GAL or GUS enzymes, respectively, resulting in fluorescent precipitates and non-fluorescent cleaved products.
However, one disadvantage of known fluorogenic substrates is their impermeability for cell membranes. As a consequence, GUS and GAL assays are generally destructive for the cells, i.e. the cell membrane needs to be permeabilized prior to detection of an intracellular analyte, and are therefore not suitable for use under in vivo conditions, or under in vitro conditions where cell integrity is desired. Many other substrates are in contrast not sufficiently photostable, such as fluorescein, which bleaches after a few minutes, loosing the fluorescence necessary for detection.
There exist in the state of the art various methods to introduce a membrane-impermeable substance into a cell. One approach is to micro inject the molecules into the cell through a micropipette. Other approaches consist of partially disrupting the structure of the cell plasma membrane, by using a powerful electric shock or a chemical such as a low concentration of detergent. A third method for introducing large molecules into cells is to cause membranous vesicles containing these molecules to fuse with the cell plasma membrane.
As a consequence, the present invention aims to provide a new approach for introducing a membrane-impermeable fluorogenic substrate into a cell. In particular, it is an object of present invention to provide novel fluorogenic hydrolase substrates, which have an increased permeability for cell membranes without jeopardising their photostability, and which are specifically recognised by specific enzymes.
Further, it is also an object of the present invention to provide methods for preparing the subject fluorogenic hydrolase substrates, substantially free of impurities.
Present invention also aims to provide fluorogenic hydrolase substrates, which can be used in a variety of cellular assays. In particular the subject fluorogenic enzyme substrates can be used in different applications, such as for studying or detecting various characteristics related to enzyme activity, gene expression or promoter activity and specificity.